The production, interconversion, and transfer of C1 units is an important basic metabolic system in all of biology. C1 units are required for a number of biosynthetic reactions and in addition, formaldehyde is produced in a number of metabolic reactions and must be detoxified. Methylotrophs are microorganisms capable of growth on C1 compounds as sole carbon and energy sources, and methylotrophy can be viewed as a specialized version of the C1 metabolism found in all organisms. We have used the genome sequence of a model methylotroph, Methylobacterium extorquens AM1 generated in this project as a platform to develop a new conceptual framework for how assimilatory and energy metabolism achieve dynamic balance in this organism. We propose to begin to test this model using a combination of biochemical, genetic, genomic, and modeling approaches. First, we will determine the remaining unknown details of a key part of assimilatory metabolism, the glyoxylate regeneration cycle, especially with respect to the steps generating reducing equivalents. This aim addresses the question of how reducing equivalents are balanced during methylotrophic growth. Second, we will assess small molecule regulators of the 13-hydroxybutyryI-CoAbranchpoint between two of the main assimilatory pathways, the glyoxylate regeneration cycle and PHB synthesis. This aim addresses the question of what signals are involved in regulating the flow of carbon at the main assimilatory branchpoint. Finally, we will assess expression of the genes involved in the three main assimilatory pathways at both transcript and enzyme activity levels and correlate this information with alterations in cellular pools of key intermediates and cofactors. This aim addresses the question of how carbon flow is balanced between these pathways according to cell needs. The result of this study will be a systems-level understanding of central assimilatory metabolism in methylotrophy. These approaches will provide a model for functional genomics at the physiological level, and will create a platform for future studies of the integration of methylotrophic assimilatory and energy metabolism and the switch between methylotrophy and heterotrophy.